Human papillomavirus (HPV)-16 DNA is frequently found in cervical cancer and precancerous lesions. Progression to invasive cervical carcinoma is associated with the integration of HPV-16 DNA fragments in the cellular genome. The integrated HPV fragments preserve and express the candidate viral oncogenes, E6-E7, but the viral E2 regulatory gene region is disrupted or inactivated. To help understand the functional consequences of HPV-16 integration seen in malignant progression, we propose to explore the interactions between cellular and viral regulatory factors at the transcriptional promoter of the candidate E6-E7 oncogene region of HPV-16, P97, in transfection and DNA binding studies. Based on our recent data (Cripe et aL, 1987, ref. 1), the HPV-16 P97 promoter contains a cis enhancer activated by cellular trans-acting factors in uninfected human genital keratinocytes and cervical carcinoma cells, but not in human or animal fibroblasts. The HPV-16 enhancer response to cell factors can be suppressed by a repressor encoded in the E2 gene region, suggesting that E2 disruption in a random integration event may release the viral E6-E7 oncogenes from viral E2 control. We will (i) map the critical cis elements of the HPV-16 cis enhancer in transfection and DNA binding assays; (ii) determine whether the HPV-16 P97 cis enhancer and homologous cis sequences of cytokeratin gene promoters respond to related cellular factors; and (iii) study how known viral regulatory factors modulate HPV-16 P97 enhancer activity in transfected cells, and whether they influence cell factor binding. The proposal is based on procedures established in the laboratory, on additional preliminary data verifying the feasibility of the experiments, and the availability of multiple molecular mutants in the viral cis sequences and in the viral regulatory genes. In this way, we hope to learn how these critical regulatory factors influence expression of the HPV-16 E6-E7 oncogene region, and thus help understand the molecular events which ultimately result in the malignant progression of HPV-16-infected cervical epithelial cells.